In response to Bioterrorism threats it has become increasingly important to perform rapid and precise detection of biological warfare agents. The Bacillus anthracis bacterium is a member of the endospore forming Bacillus cereus group. Bacillus anthracis is a highly lethal biological warfare agent that is easy to obtain, store, and apply as a bioweapon. In order to make a reliable detection of Bacillus anthracis the DNA must be analyzed, since phenotypic differences between the members of the Bacillus cereus group in some instances are is less than 1%.
As an example, Bacillus cereus differs only from Bacillus anthracis because the latter contains two additional plasmids called pXO1 and pXO2. Avirulent strains of Bacillus anthracis lacking pXO1 and pXO2 are virtually indistinguishable from Bacillus cereus. Theoretically, transfer of the pXO1 and pXO2 plasmids into members of the Bacillus cereus group will turn these bacteria into functional Bacillus anthracis. For this reason DNA analysis and discrimination of the plasmids pXO1 and pXO2 by means of DNA hybridization, sequencing, or PCR is the only valid method for determining if the detected organism is Bacillus anthracis. 
Due to their capability to resist harsh environments, the liberation and extraction of DNA from an endospore is a difficult task. The normal procedure used in the detection of Bacillus anthracis is to germinate the spores in a culture substrate, collect the bacteria and subsequently extract the DNA from the vegetative bacteria, a procedure that can take many hours up to a day (Levi et al 2003). Other methods include elaborate techniques like mechanical disruption, freeze/thaw cycles or chemical treatment (Johns et al 1994). However, the spore coat and cortex are biochemical structures developed for long term hibernation that can last thousands of years. A famous example is a bacterium revived from an endospore found in the gut of a bee embedded in amber (Cano & Borucki 1995). Furthermore, mechanical disruption (bead beating) results in poor quality of the released DNA (Levi et al 2003). Using present technology, it is possible to release DNA within 5-10 minutes from endospores by combing physical, mechanical, and chemical treatment, but even 5 minutes for DNA extraction is considered long time when the application is a monitoring of bioterrorism attacks carried by aerosols. The use of elaborate multistep procedures is not optimal in the stressful situation that a possible anthrax attack is. For this reason there is a need for a technology that allows rapid (within seconds) hands-off single step DNA extraction from endospores of Gram positive bacteria.
Gram positive bacteria of the genus Bacillus and Clostridia are capable of undergoing a process at the end of the exponential growth phase called sporulation. During sporulation the bacteria form a rugged spore capable of persisting harsh environments. The spore is a dormant structure with only a few metabolic active enzymes that induced germination when the spore is exposed to nutrients. The spore is very different in its biochemical composition as depicted in Table 1.
TABLE 1Differences in biochemical composition existing between sporesand vegetative cells of Bacillus species.Levels of molecules (μmol/g [dry weight]Small moleculeBacillus sporeBacillus vegetative cellNADH<0.0021.95NAD0.110.35NADPH<0.0010.52NADP<0.0180.44ATP<0.0053.6ADP0.21AMP1.2-1.313PGA 5-18<0.2DPA410-470<0.1Ca2+380-916Mg2+ 86-120Mn2+27-56H+6.3-6.57.5-8.2
The biochemical structures and the dormant physiological state makes the endospore an extremely mechanical, chemical, and heat resistant entity that poses a particular problem in terms of rapid sample preparation and DNA extraction of biological warfare agents for rapid identification. The spores can resist e.g. prolonged boiling without breaking apart. The environmental fate of the spore is not known in detail. The spores can survive ‘indefinitely’ in dry and protected environments. Excavations in Kruger National Park in South Africa revealed B. anthracis spores more than 200 years old (as dated by the 14C method) that were still able to germinate in the laboratory.
The most sensitive methods of detecting bacteria and vira rely on gaining access to the intracellular components of the organisms, such as their genetic material.
US 2003/0,146,100 discloses dielectrophoretic separation of cells from blood followed by electronic lysis on isolated cells and digestion, performed on one chip with an electrode array in a flow chamber. Electrophoresis performed with 10 KHz sinusodial field and lysis performed with a series of 400 pulses of 500V and 50 μs duration or 40 pulses of 200V and 10 μs duration (square wave).
US 2002/0115201 discloses cell lysis using microwaves in the range of 2.45-310 GHz thereby releasing DNA from cells in liquid suspension. It furthermore discloses a chamber with parallel planar external electrodes for applying microwaves, the chamber have flow channels for providing sample in chamber.
U.S. Pat. No. 4,970,154 discloses a method for inserting foreign genes into cells using pulsed radiofrequency. It furthermore describes electroporation and fusion of cells suspended in a solution in a chamber using pulsed radiofrequency oscillating electrical fields between electrodes in the chamber. Frequency varies from 60 Hz to 10 MHz, fields strength of 100-400 V/cm.